There is a plethora of approaches to arranging for controlled release of drugs or growth factors useful in medicine. For example, compositions and methods have been described for controlled release of drugs covalently coupled to polyethylene glycol (PEG). As opposed to a drug bound irreversibly to PEG in order to enhance its half-life and diminish its immunogenicity, conjugates have been prepared wherein PEG is a releasable carrier of the drug or prodrug. Typically, the drug is attached by an ester or carbonate linkage that can be cleaved by esterase-catalyzed hydrolysis. The adjustment of release rates in these cases is, however, difficult. Examples are PEG-camptothecin, PEG-SN38, PEG-irinotecan and PEG-docetaxel. Additional adaptations have been made to accommodate amine-containing drugs whereby a PEG moiety is connected by a cleavable ester to a self-immolating carbamate. This technology has been applied to peptides and proteins as well as to daunorubicin, amphotericin, Ara-C and other small molecules.
Another system has been developed at the Weizmann Institute wherein PEG or other macromolecule is attached to beta elimination linkers such as fluorenyl methoxycarbonyl (Fmoc) or its 2-sulfo derivative (Fms). These are described in U.S. Pat. No. 7,585,837. However, rate-of-release control remains a problem.
PCT publication WO2009/158668 describes releasable drug conjugates to macromolecules wherein the rate of beta elimination is controlled by a trigger independent of the link to the macromolecule itself. This solves a problem left unsolved in the prior art.
The release mechanism set forth in the '668 PCT publication has not been applied to instances where a multiplicity of drug molecules is coupled covalently, but releasably, to dendrimeric macromolecules. It is also limited to drugs that contain an amine functional group. In addition to providing a controllable rate of release of more than one drug molecule from the dendrimer itself, this approach offers a means whereby the coupled drug is protected from hydrolysis by the presence of a protective polymer, such as PEG, on different sites at the surface or interstices of the solid support.
Dendrimers have been used as carriers for therapeutic compounds, either by entrapment of a drug in cavities within the dendrimer, or by covalently linking drug molecules to the surface. This is reviewed in Svenson, S., Eur J Pharm Biopharm (2009) 71:445-462 and Cheng, Y., J. Pharm. Sci. (2007) 97:123-143. Entrapment within dendrimer cavities is limited to small molecules, and covalent attachment approaches have thus far been limited to systems in which a small drug is hydrolytically or enzymatically cleaved from the dendrimer surface. Unmodified cationic dendrimers such as polyamidoamine dendrimer (PAMAM) or poly-L-lysine (PLL) have biocompatibility and toxicology shortcomings, for example, disruption of cell membranes and also have very short half-lives, typically <20 min. Toxicity may be reduced by functionalizing the surface of the dendrimer with non-ionic or anionic groups (Kaminskas, L., et al., Mol Pharm (2007) 4:949-961). PAMAM dendrimers are not biodegradable and are retained in the liver and kidney, raising a concern—albeit unproven—of toxicity upon chronic dosing, though PLL dendrimers while retained in the liver and kidney, appear to be broken down to constituent monomers.
It has been shown that PEGylation of PAMAM and PLL dendrimers neutralizes the surface positive charges and reduces or eliminates their propensity to lyse cells and cause acute toxicity. It has also been shown that the hydrophilic PEG moiety increases water solubility of guest drug molecules, and that PEGylated dendrimers effectively accumulate in tumor tissue via the enhanced permeability and retention and thus serve as targeting delivery vehicles for anti-tumor agents. PEGylated PLL shows almost complete (>90%) bioavailability when administered subcutaneously, providing downstream benefit in terms of patient compliance. Most importantly, PEGylation of cationic dendrimers can decrease renal filtration and dramatically increase the half-life from minutes to several days. Composite results show that long half-lives may be achieved with PEGylated dendrimers of MW≧40 kDa by varying either the number or size of the PEG chain. That is, the MW of the total dendrimer-PEG conjugate rather than the dendrimer or individual PEG chains dictates the extent of renal filtration. The size of PEGylated poly L-lysine dendrimer complexes can be specifically manipulated to dictate their pharmacokinetics, biodegradation and bioresorption behavior.
In particular, camptothecin attached to the dendrimer surface of PLL16(PEG5000)8 (i.e., a PLL with 16 functional groups at the surface, 8 of which are occupied by PEG of 5000 molecular weight) via an ester linker was completely protected from serum esterases, whereas an analogous PEG-camptothecin ester hydrolyzed ˜10-fold faster in serum than buffer. A tetra-peptide chymotrypsin substrate attached to the dendrimer end groups of PAMAM32(PEG2000)20 was protected against chymotrypsin hydrolysis (kcat/KM 0.1 uM−1 s−1) compared to the peptide-dendrimer without PEG (˜5-fold), or to the free peptide (˜8-fold), or to the peptide attached to PEG (˜12-fold).* IFN-α-2b attached to the core of a 4-armed PEGylated-dendrimer showed ˜10-fold lower cytotoxicity and anti-viral activity, as well as trypsin resistance and prolonged serum half-life compared to the free cytokine. Thus, it appears that molecules covalently bound to a dendrimer core and immersed within a PEG layer are protected against hydrolytic enzymes. * The rate of diffusion of chymotrypsin into the PEG shell is kcat/KM; so the results indicate that proteins of at least this size (25 kDa) should as well escape out of the PEG layer.
The compositions of the invention overcome problems associated with coupling drugs to the conventional monovalent linear PEG carrier. In order to minimize kidney filtration, the molecular weight of the PEG carrier must be at least about 40,000 and the drug is limited therefore to about 1 μmole per 40 mg PEG. Thus, only very potent drugs can employ this system as a practical matter. Linear PEG's also provide only limited protection against enzymes that may modify and/or destroy the bound drug. Drugs bound to linear PEG may retain significant biological activity; while this is a requirement for a stably-modified drug, use of PEGylated-drug as a carrier for slow release of active free drug requires that the PEGylated form be substantially inactive due to the relatively higher dosages involved. The present invention permits increasing the drug payload and protects the drug against degrading enzymes as well as blocking access of the drug to its biological receptor. Like the technology described in the '668 publication, the activity of the drug is silenced until it is released, permitting administration of relatively large doses as depots.